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The object of this study can be found among the various ornamental rocks used in historic buildings in the city of Rio de Janeiro. It is a degraded Leptinito Gneiss that makes up one of the support columns of the kitchen of the Mosteiro de São Bento (Monastery of Saint Benedict) in Rio de Janeiro. The main aim of the present study is to identify the causes of the high degree of degradation of said column. Non-destructive tests were performed, and laboratory evaluation of the disintegrated fragments may help restore and conserve this column in the future. Results obtained from the tests performed on the altered column were compared to those obtained from another column in the monastery, also built in Leptinito, which is more intact and is a sound Leptinito Gneiss. The results showed that degradation of the column is caused by the crystallization of salts (halite) inside the rock, which is reducing its mechanical strength and causing an imminent risk of collapse.

This article aims to highlight stroke considerations in the evaluation and management of dysphagia. Although dysphagia was previously thought to occur only following brainstem or bilateral cortical strokes, the development of brain imaging and dynamic swallowing studies has revealed small, unilateral supratentorial strokes can produce dysphagia. In this article, screening, evaluation, and management of dysphagia are outlined, as well as the clinical decision making that occurs when taking into account cognitive and communication deficits that may be present. For the clinical swallow examination, chart review, interview, informal evaluation of cognition and communication, observation of posture, oral cavity inspection, cranial nerve examination, and the direct swallowing assessment are reviewed along with tailoring of each according to the deficits observed. Specific compensation and rehabilitation strategies are discussed along with how cognitive and communication deficits can guide the clinician's decision-making process to select an appropriate plan of care. A case study is provided to synthesize the process into a real-world scenario., Competing Interests: Disclosure The authors report no conflicts of interest in this work., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

The objective of this study is to get to know the nursing students' perception in terms of the physical, educational and human aspects of the nursing laboratory in the teaching-learning process. This is a quantitative, cross-sectional, non-experimental study. Participants were 85 students who answered to a questionnaire about the laboratory in regard to human resources, teaching resources, physical infrastructure, and accommodation and hours of operation. Regarding the physical infrastructure of the lab, the majority of students (58.8%) rated it as bad or average, and accommodation was rated between bad and extremely bad by 50.6% of the students. The lab working hours were rated from good to excellent by 63.5% of the students. The nursing specialists and monitors of the laboratory were positively rated by the students, with 87.0% and 84.9% rating them from good to excellent, respectively. According to the students, the best aspect of the laboratory was human resources.

The purpose of this study was to compare the execution and verify the compliance with the handwashing techniques by students in an undergraduate Nursing course. The study was performed with 113 students enrolled in internship programs in healthcare institutions in the city of São Paulo. Data collection occurred through direct observation, using a check-list instrument with the steps of the technique. Students in their 2nd and 3rd year had better results in most steps of the technique, with a statistically significant difference when compared to the 4th year. Compliance with the steps of the technique by the students was very low, since their compliance with half the handwashing steps was lower than 50%. The average amount of students who executed all the steps of the technique correctly was very low, 8.8%. The observed students did not execute the handwashing techniques according to the recommendations.

Background: Anti-oxidant vitamins have increasingly been used to supplement traditional post-surgical treatment in cardiac transplant recipients. However, the mechanism(s) of action have not been determined. In this study we examined the effects of a novel vitamin E analog, alpha-tocopheryl polyethylene glycol-100 succinate (alpha-TPGS), and low-dose cyclosporine (CsA) in the treatment of acute and delayed cardiac rejection., Methods: In situ sonomicrometry, histologic rejection and graft survival were determined in untreated rat cardiac allograft recipients and recipients receiving CsA, alpha-TPGS or CsA plus alpha-TPGS. DNA binding of nuclear factor (NF)-kappaB and AP-1, inducible nitric oxide synthase (iNOS) protein, caspase-3 activity and lymphocyte proliferation were determined., Results: alpha-TPGS significantly (p < 0.05) prolonged graft survival equipotent to low-dose CsA. Treatment with CsA plus alpha-TPGS further enhanced graft survival (p < 0.001). CsA or alpha-TPGS alone decreased rejection, with the greatest decrease seen using combination therapy. Graft fractional shortening was improved by CsA or alpha-TPGS alone (p < 0.01), whereas distention in systolic and diastolic lengths in untreated allografts was prevented by CsA, alpha-TPGS and combination therapy. Nitrosylation of heme protein was inhibited by alpha-TPGS and abolished by CsA or CsA plus alpha-TPGS. Expression of iNOS was decreased 50% by alpha-TPGS equipotent to CsA, but apparently via an NF-kappaB- and AP-1-independent pathway. Caspase-3 activity, an index of apoptosis, was increased only in untreated allografts. In addition, alpha-TPGS markedly inhibited mitogen-stimulated proliferation by both rat and human lymphocytes., Conclusions: alpha-TPGS has a significant effect in limiting lymphocyte proliferation and activation. This might explain the equipotent action of alpha-TPGS vs low-dose CsA and its action to potentiate graft survival and limit graft rejection and dysfunction.

Measurement of the bimolecular collision rate between a spin label and oxygen is conveniently carried out using a gas permeable plastic sample tube of small diameter that fits a loop-gap resonator. It is often desirable to concentrate the sample by centrifugation in order to improve the signal-to-noise ratio (SNR), but the deformable nature of small plastic sample tubes presents technical problems. Solutions to these problems are described. Two geometries were considered: (i) a methylpentene polymer, TPX, from Mitsui Chemicals, at X-band and (ii) Teflon tubing with 0.075 mm wall thickness at Q-band. Sample holders were fabricated from Delrin that fit the Eppendorf microcentrifuge tubes and support the sample capillaries. For TPX, pressure of the sealant at the end of the sample tube against the Delrin sample holder provided an adequate seal. For Teflon, the holder permitted introduction of water around the tube in order to equalize pressures across the sealant during centrifugation. Typically, the SNR was improved by a factor of five to eight. Oxygen accessibility applications in site-directed spin labeling studies are discussed.

Spin-trapping nitrones such as alpha-phenyl-N-tert-butylnitrone (PBN) have traditionally been used to trap and stabilize free radicals for detection by electron paramagnetic resonance (EPR) spectroscopy. Unlike classical antioxidants, these agents have never been evaluated therapeutically in allograft transplantation. In the present study, we examined potential mechanisms of action of treatment with PBN in a rat model of acute cardiac allograft transplantation. Graft rejection was determined by histological examination and graft function determined by in situ sonomicrometry. DNA binding for nuclear factor (NF)-kappaB and activator protein (AP-1) were determined by gel shift assays. Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed for inducible nitric-oxide synthase (iNOS) and inflammatory cytokines. Histological rejection scores were elevated in untreated allografts and decreased by treatment with PBN. In situ sonomicrometry revealed decreased heart rate and distended end diastolic and end systolic segment lengths with rejection. Although PBN did not alter heart rate, it did normalize the distention of both diastolic and systolic cardiac dimension. EPR spectroscopy revealed nitrosylation of myocardial heme protein in untreated allografts that was decreased by treatment with PBN. PBN also decreased iNOS protein and iNOS mRNA. RT-PCR analysis revealed enhanced cytokine gene expression for interferon-gamma, interleukin-6, and interleukin-10 in untreated allografts. Expression for these genes was potently inhibited or abolished in recipients treated with PBN. PBN treatment also decreased DNA binding of transcription factors, NF-kappaB and AP-1. Thus, PBN retains significant anti-inflammatory properties through its action to down-regulate cytokine gene expression that contribute to protection against acute alloimmune activation in cardiac allografts.

Dithiocarbamate derivatives sequester metals such as iron and may have benefits in inflammatory diseases. We examined the actions of a new dithiocarbamate-based oral formulation, NOX-700, on protein modification by nitric oxide (NO), gene expression, and lymphocyte proliferation in a model of acute and delayed cardiac rejection. Chronic treatment with NOX-700 prolonged graft survival. In combination with low-dose cyclosporine (CsA), NOX-700 produced a synergistic action to prolong graft survival. NOX-700 decreased myocardial heme nitrosylation. A single bolus injection with NOX-700 in untreated recipients did not decrease heme nitrosylation but normalized NO metabolites and caused the formation of a mononitrosyl iron complex indicating NO scavenging in vivo. NOX-700 alone given with CsA inhibited protein nitration. NOX-700 or CsA each alone decreased intragraft inflammatory cell infiltration. NOX-700 also potentiated the CsA-induced inhibition of splenocyte proliferation ex vivo stimulated by concanavalin A. In splenocytes derived from treated rats but stimulated ex vivo in a mixed lymphocyte response (MLR), interferon-gamma and cyclin D3 gene expression was inhibited by NOX-700 suggesting down-regulation of lymphocyte activation and proliferation by in vivo treatment. These studies suggest that NOX-700 is protective in cardiac rejection, in part, by scavenging of NO and by limiting lymphocyte activation infiltration.

We examined the efficacy and mechanism of action of N(6)-(1-iminoethyl)-L-lysine (L-NIL), a highly selective inhibitor of inducible nitric oxide (NO) synthase (iNOS), on acute cardiac transplant rejection. L-NIL produced a concentration-dependent attenuation of plasma NO by-products and a decrease in nitrosylation of heme protein without altering protein levels of iNOS. At postoperative day 4, L-NIL did not alter the increased binding activities for transcription factors nuclear factor-kappaB and activator protein-1. Whereas L-NIL decreased inflammatory cell infiltration, graft survival was only prolonged at the dose of 1.0 microg/ml that incompletely blocked NO production. Higher L-NIL concentrations (30 and 60 microg/ml) ablated the increased NO production but failed to improve graft survival and even potentiated NF-kappaB binding activity examined at day 6. Alloimmune activation indicated by increased cytokine gene expression for interferon-gamma, interleukin-6, and interleukin-10 was inhibited in grafts only by treatment with 1.0 microg/ml L-NIL. These findings suggest a complex role of NO in acute cardiac allograft rejection. Partial inhibition of iNOS is beneficial to graft survival, whereas total ablation may oppose any benefits to graft survival. These studies have important implications in understanding the dual role of NO in acute rejection and help to reconcile discrepancies in the literature.

In this study, we examined the actions of diethyldithiocarbamate-iron (DETC-Fe) complex in acute graft rejection heterotopically transplanted rat hearts. Chronic treatment with DETC-Fe inhibited the increase in plasma nitric oxide (NO) metabolites and nitrosylation of myocardial heme protein as determined by electron paramagnetic resonance (EPR) spectroscopy. Pulse injection with DETC-Fe normalized NO metabolites. We verified intragraft trapping of NO in vivo by pulse injection with DETC-Fe by the detection within allografts of an anisotropic triplet EPR signal for DETC-Fe-NO adduct with resonance positions (g tensor factors for perpendicular and parallel components, respectively g( perpendicular ) = 2.038 and g( parallel ) = 2.02; hyperfine coupling of 12.5 G). DETC-Fe prolonged graft survival and decreased histological rejection scores. DNA binding activity for nuclear factor (NF)-kappaB and activator protein-1 was increased in allografts and prevented by DETC-Fe. Abrogation of the activation of NF-kappaB by DETC-Fe was associated with increased IkappaBalpha inhibitory protein. Western blotting and RT-PCR analysis revealed that DETC-Fe inhibited inducible NO synthase protein and gene expression. Gene expression for the proinflammatory cytokine interferon-gamma was also decreased by DETC-Fe. Thus DETC-Fe limits NF-kappaB-dependent gene expression and possesses significant immunosuppressive properties.

We examined iron nitrosylation of non-heme protein and enzymatic activity of the Fe-S cluster protein, aconitase, in acute cardiac allograft rejection. Heterotopic transplantation of donor hearts was performed in histocompatibility matched (isografts: Lewis --> Lewis) and mismatched (allografts: Wistar-Furth --> Lewis) rats. On postoperative days (POD) 4-6, Western blot analysis and immunohistochemistry revealed inducible nitric-oxide synthase (iNOS) protein in allografts but not isografts. EPR spectroscopy revealed background signals at g = 2.003 (for semiquinone) and g = 2.02 and g = 1.94 (for Fe-S cluster protein) in isografts and normal hearts. In contrast, in allografts on POD4, a new axial signal at g = 2.04 and g = 2.02 appeared that was attributed to the dinitrosyl-iron complex formed by nitrosylation of non-heme protein. Appearance of this signal occurred at or before significant nitrosylation of heme protein. Iron nitrosylation of non-heme protein was coincidental with decreases in the nonnitrosylated Fe-S cluster signal at g = 1.94. Aconitase enzyme activity was decreased to approximately 50% of that observed in isograft controls by POD4. Treatment with cyclosporine blocked the (i) elevation of plasma nitrate + nitrite, (ii) up-regulation of iNOS protein, (iii) decrease in Fe-S cluster EPR signal, (iv) formation of dinitrosyl-iron complexes, and (v) loss of aconitase enzyme activity. Formation of dinitrosyl-iron complexes and loss of aconitase activity within allografts also was inhibited by treatment of recipients with a selective iNOS inhibitor, l-N(6)-(1-iminoethyl)lysine. This report shows targeting of an important non-heme Fe-S cluster protein in acute solid organ transplant rejection.

The purpose of this work was to improve our current knowledge of the non-enzymatic mechanisms involved in brown rot fungal decay of wood, but also to improve our understanding of chelator-mediated reactions that may occur in other biological systems where low pH conditions may prevail. Several key steps (iron chelation and reduction) during early non-enzymatic wood decay processes have been studied by using electron spin resonance (ESR). It has been proposed that low molecular weight chelators as well as Fenton reagents are involved in brown rot decay, at least in early non-enzymatic stages. In this work, the binding between a catecholate model chelator and ferric iron was studied by ESR spectroscopy. The effects of the model chelator, Fenton reagents, as well as the reaction conditions on free radical generation were also studied using ESR spin-trapping techniques. The results indicate: (1) The relative quantity of the chelator-iron complex can be determined by measurement of the intensities of the characteristic g = 4.3 ESR signal. (2) The effects of the chelator:iron ratio, the pH, and other reaction parameters on the hydroxyl radical generation in a Fenton type system can be determined using ESR spin-trapping techniques. (3) Data support the hypothesis that superoxide radicals are involved in chelator-mediated Fenton processes.

Background: Defective endothelium-dependent relaxation is observed in experimental and human diabetes mellitus. The nature of this defect is not fully understood but may involve decreased nitric oxide (NO) bioactivity due to enhanced production of reactive oxygen species (ROS). In this paper, we examine the benefits and actions of a novel NO-donating, antioxidant called 2-acetoxybenzoic acid 2-(2-nitrooxymethyl) phenyl ester, and denoted as NCX4016, on NO-mediated endothelium-dependent relaxation in normal arteries exposed to acute elevations in glucose or in arteries derived from chronic diabetic animals., Material and Methods: Intrinsic free radical scavenging by NO-NSAIDs in solution were evaluated using electron paramagnetic resonance (EPR) spectroscopy and spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). In acute studies, normal rat aortas were exposed in tissue culture for 18 h to 5.5 mM or 40 mM in the presence or absence of NCX4016, a NO-donating NSAID unrelated to aspirin (NCX2216) or aspirin. Vascular reactivity of thoracic aortic rings to endothelium-dependent relaxation to acetylcholine in vitro was determined. For chronic hyperglycemia, diabetes was induced in rats by intravenous injection with streptozotocin. Vascular reactivity of thoracic aortic rings to endothelium-dependent relaxation to acetylcholine in vitro was determined after 8 wks in untreated animals or animals chronically-treated with NCX4016. Antioxidant efficacy in vivo was determined by measurement of plasma isoprostanes and by nuclear binding activity of NF-kappaB in nuclear fractions of aortae., Results: Incubation with NCX4016 and NCX2216 produced a concentration-dependent inhibition of DMPO-OH formation indicating scavenging of hydroxyl radicals (HO(*)). In contrast, little efficacy to scavenge superoxide anion radicals was noted. Acute incubation of normal arteries with elevated glucose concentration caused inhibition of normal relaxation to acetylcholine. This impairment was prevented by co-incubation with NCX4106 but not by mannitol, the parent compound (aspirin) or by NCX2216. In addition, chronic treatment with NCX4016 prevented the development of defective endothelium-dependent relaxation to acetylcholine. This protection did not occur as a result to any changes in blood glucose concentration or hemoglobin glycation. Treatment with NCX4016 did decrease the elevation in plasma isoprostanes and normalized the diabetes-induced increase in NF-kappaB binding activity in nuclear fractions derived from aortic tissue., Conclusions: Collectively, these studies suggest that antioxidant interventions using NO-donating NSAIDs may provide an important novel therapeutic strategy to protect the diabetic endothelium.

Nitric oxide (NO) derived from the up-regulation of inducible NO synthase (iNOS) is believed to play an important role in organ rejection. In experimental models of acute cardiac transplant rejection (i.e., without immunosuppression), treatment using NOS inhibitors to prevent acute rejection have yielded conflicting results. This is most likely due to potential inhibition of constitutive NOS. Accordingly, agents that trap NO directly may have some advantage. In the current study, we evaluated the actions of a ruthenium-based NO scavenger, AMD6221, to inhibit the nitrosylation of myocardial protein and to prolong cardiac allograft survival in a model of acute cardiac transplant rejection (without immunosuppression). In addition, we evaluated the efficacy of AMD6221 used in combination with low-dose cyclosporine (CsA) (i.e., a model of delayed graft rejection). Heterotopic abdominal cardiac transplantation was performed using rat strains with disparities at major and minor histocompatibility loci. Grafts were harvested on postoperative day 6 for histologic examination or analysis of myocardial protein nitrosylation using electron paramagnetic resonance (EPR) spectroscopy. Other animals were monitored twice daily to determine rejection times. Plasma was also taken at postoperative day 6 for determining the concentration of NO by-products (nitrate plus nitrite). Treatment with AMD6221 either prolonged graft survival and/or caused a marked decrease in myocardial nitrosylprotein formation as determined by EPR spectroscopy. In vivo scavenging of NO by AMD6221 was verified by high-performance liquid chromatography analysis of nitrosylated-drug in plasma samples. Low-dose CsA given alone or in combination with AMD6221 completely blocked formation of myocardial nitrosylprotein complexes. Whereas low-dose CsA alone prolonged graft survival, combination therapy with CsA plus AMD6221 produced a synergistic effect on graft survival. These studies indicate that treatment with a ruthenium-based NO scavenger, such as AMD6221, may be an effective regimen used alone or in combination with CsA to protect myocardial proteins from posttranscriptional modification and to prolong cardiac graft survival.

Nitric oxide synthase (NOS) inhibitors have been shown to reduce NO but yield conflicting results on cardiac allograft survival. In this study, we provide an alternative approach specifically to examine the efficacy of a NO scavenger on nitrosyl complex formation and graft survival in a model of heterotopic cardiac transplantation. Efficacy was examined under both acute and chronic conditions (i.e., without or with immunosuppression, respectively). Electron paramagnetic resonance (EPR) spectroscopy of frozen myocardial tissue from untreated allografts showed progressive increases in nitrosylheme and nitrosomyoglobin before graft failure. These signals were not seen in either isografts or native hearts of allograft recipients. Both plasma nitrate plus nitrite and myocardial nitrosyl complex formation in cardiac allografts were significantly decreased in recipient animals treated with the NO scavenger, NOX-100, or by low-dose cyclosporine (CsA). Both interventions were nearly equivalent in significantly prolonging graft survival. The short-term combination treatment of both NOX-100 plus CsA completely eliminated myocardial nitrosyl complex formation and synergistically prolonged graft survival. Long-term combination drug treatment (days 0-100) followed by cessation of therapy resulted in permanent graft acceptance with no evidence for nitrosyl complex formation. These studies support a role of NO in cardiac allograft rejection. Furthermore, these studies indicate a potential therapeutic value of NO scavengers in preventing organ rejection.

Thiyl radicals (RS.) formed during peroxynitrite- or hydroxyl radical-dependent oxidation of thiols, i.e., glutathione (GSH) and L-cysteine (CySH) were trapped with 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) and analyzed by X-band and Q-band electron spin resonance (ESR) spectroscopy. At X-band, the ESR parameters of DMPO-glutathionyl radical adduct (DMPO/.SG) and DMPO-hydroxyl radical adduct (DMPO/.OH) are nearly similar in aqueous solutions and as a result, except for the lowfield spectral line, the remaining spectral lines of DMPO/ .SG virtually over-lap with those of the DMPO/.OH adduct. In contrast, at Q-band, most of the spectral lines due to the DMPO/.SG were separated from the DMPO/ .OH. Inclusion of a superoxide dismutase (SOD) mimic completely abolished the formation of the DMPO/.OH adduct and not the DMPO/.SG adduct during ONOO(-)-mediated oxidation of GSH and DMPO. In the presence of formate, the DMPO/.SG spectrum was replaced by the DMPO/.CO2- spectrum which was monitored by Q-band ESR spectroscopy. Thus, spin-trapping at Q-band provides unambiguous proof for the glutathionyl radical-dependent oxidation of formate by peroxynitrite. High resolution Q-band ESR spectra of DMPO/.Scys were also obtained. Biological applications of the Q-band spin-trapping technique to detect thiyl radicals in cellular systems are discussed.

Electron spin resonance (ESR) spectroscopy together with spin trapping techniques and the application of state-of-the-art loop gap resonators was used to provide a direct measure of spontaneous oxygen radical production by homogenates of freshly isolated and cultured rat pancreatic islets. Using the spin trap agent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), we were able to detect production by islets of an ESR-sensitive radical signal consisting of a quartet with intensity ratio of 1:2:2:1 and hyperfine splitting of aN = aH = 14.9 Gauss, which is consistent with the DMPO-OH adduct. The amplitude of the signal was decreased by decreasing amount of islets and not detected in the absence of islets. Formation of the DMPO-OH adduct was diminished by the hydroxyl radical scavengers (e.g., ethanol, dimethylsulfoxide, and dimethylthiourea). Only partial attenuation of signal was produced by incubation with an iron chelator or using chelex-treated buffers. The ESR signal was insensitive to the xanthine oxidase inhibitor, oxypurinol, or to superoxide dismutase, but was eliminated in a concentration-dependent manner by either potassium cyanide or catalase (but not heat-inactivated catalase). These observations suggest that the origin of the DMPO-OH arose not from free hydroxyl radicals but primarily from endogenous hydrogen peroxide production perhaps of mitochondrial origin. The development of this technology has implications for the potential measure of oxygen radical production in islet homogenates under pathologic conditions as well as to the application of other cell culture systems.

This work demonstrates the use of multiquantum EPR to study the magnetic properties of copper complexes and copper proteins. Pure absorption spectra are obtained because of the absence of field modulation. The signal intensity of 3-quantum spectra is proportional to the spin lattice relaxation time T1, while its linewidth in a frequency difference sweep is T1(-1). A change in lineshape for the EPR detectable mixed value [Cu(1.5) . . . Cu(1.5)] site in nitrous oxide reductase is attributed to suppression of the forbidden transitions. The data confirm the unusually fast relaxation time for this site, which requires temperatures of less than 100 K to resolve hyperfine structure. The T1's for the mixed valence [Cu(1.5) . . . Cu(1.5)] site in nitrous oxide reductase are very similar to T1's for the Cua site in cytochrome c oxidase. The similar relaxation properties, together with previous multifrequency EPR results, support the hypothesis that the EPR detectable sites in cytochrome c oxidase and nitrous oxide reductase are mixed valence [Cu(1.5) . . . Cu(1.5)] configurations.

When urine samples from alkaptonuria patients are allowed to stand, they turn black, presumably owing to the oxidation of homogentisic acid to a melanin-like substance. We report the characterization of the pigments formed by polymerization of (a) the components in the urine from a patient with alkaptonuria and (b) homogentisic acid. The absorption spectra and electron spin resonance signals of these pigments are similar to those of eumelanins. Irradiation of the pigments with nitroblue tetrazolium caused reduction of the tetrazolium; this was partially inhibited by superoxide dismutase. Irradiation of Ehrlich ascites carcinoma cells with the pigments from homogentisic acid or urine caused cell lysis. Since this lysis was inhibited by catalase, we have concluded that it was mediated by H2O2. A similar pigment was also extracted from the tissue from an alkaptonuria patient. It is suggested that the degeneration of tissue in vivo may be due to the deposition of melanin-like pigments in the tissues, probably in combination with metal ions.

Optical data have shown that the neurotoxin metabolite 1-methyl-4-phenyl-2,3-dihydropyridinium undergoes one-electron oxidation/reduction in the presence of iron chelates. The activation energy for one-electron oxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium is less than that for two-electron oxidation. Horseradish peroxidase catalyzes the oxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium. Reactivity of 1-methyl-4-phenylpyridinyl radical is discussed in relation to the well-known pyridinyl radicals.

Synthetic pheomelanins from enzymic oxidation of the 3,4-dihydroxyphenylalanine (dopa) derivative 5-S-cysteinyldopa have been examined by ESR spectroscopy. These alkalisoluble polymers contain a novel kind of free radical that is spectroscopically distinct from that found in eumelanins. Delocalization of the unpaired electron onto a nitrogen atom and the ability of the radical to chelate complexing metal ions strongly suggest an o-semiquinonimine structure. The synthetic pheomelanin was compared with natural red pigments extracted from human red hair and from red chicken feathers. Spectroscopically, the chicken feather pheomelanin is almost identical to synthetic cysteinyldopa pheomelanin. In contrast, the pigment from red hair has a major spectral component very similar to that found in dopa melanin, with a smaller component corresponding to that found in cysteinyldopa melanin.

The incorporation of natural eumelanin from bovine eyes and synthetic 3,4-dihydroxy-phenylalanine (dopa) melanin into Chinese hamster ovary (CHO) cells is reported. The process is linear for at least 8 h. Electron microscopy showed phagocytosis of melanin, either as a single granule or in groups of granules, into cell lysosomes with subsequent degradation of the granule. The general features of the ingestion and degradation processes mimic those of the incorporation of melanosomes into keratinocytes. CHO cells with ingested melanin in general revealed properties very similar to those of the pigment-free CHO cell: cell division, oxygen consumption and plating efficiency were not greatly altered by moderate concentrations of pigment. This suggests that the CHO cell system may be useful for the study of pigment in a cellular environment; pigment-free CHO cells are well characterized and can serve as a good control. Preliminary applications are reported: demonstrations of (1) incorporation of metal ions (Al3+) into CHO cells using melanin as a carrier; (2) the ability of melanin to enhance the rate of oxygen consumption during photo-irradiation of the cells.

The ESR-spin stabilization approach has been employed to detect and characterize o-semiquinone radicals from the oxidation of epinephrine and related materials (norepinephrine, 3,4-dihydroxynorephedrine, isoproterenol, and adrenalone) in aqueous solutions. Semiquinones were generated by various oxidative procedures--enzymatic oxidation (with horseradish peroxidase/H2O2), chemical oxidation (with Ag2O) and photooxidation--and subsequently kinetically stabilized through complexation with Zn2+ ions. This "spin stabilization" affords high radical concentrations, which has allowed unambiguous identification of the radical intermediates. Where appropriate, spectral assignments have been supported by deuterium substitution experiments and computer simulations of spectra. Two types of free radical have been identified: primary "open chain" semiquinones, formed by one-electron oxidation of the parent catecholamines, and secondary semiquinones, formed subsequent to cyclization reactions. The mechanism of formation of the secondary radicals is discussed, and it is concluded that they are derived from product aminochromes. Thus, oxidation of adrenochrome gives a radical identical to the secondary species observed from oxidation of epinephrine.

Using spin stabilization, ESR measurements have been made of o-semiquinone production from the horseradish peroxidase-H2O2 oxidation of catecholamine substrates. The termination rate constant for semiquinones stabilized with Zn2+ at pH 5 is about 10(4) times smaller than for uncomplexed semiquinones at neutral pH. Stabilization allows steady state concentrations of semiquinones to be obtained. The duration of the steady state is dependent upon the concentrations of enzyme, hydrogen peroxide, and catecholamine substrate. The relative reactivity of the substrates 3,4-dihydroxyphenylalanine, norepinephrine, and dopamine at pH 5 is 1:8:40. The effects of phenol and ascorbate were studied and shown to be consistent with scavenging of phenoxyl radicals by catecholamine and semiquinone radicals by ascorbate, respectively.

Investigations were carried out to determine whether the melanin present in the blue and brown eyes were eumelanin, the melanin present in black hair and dark skin, or pheomelanin, the melanin present in red hair and the skin of people with red hair. Our results showed that UV-visible irradiation of blue or brown eye melanin did not produce any superoxide. Irradiation of 51Cr-labelled Ehrlich ascites carcinoma cells in the presence of blue or brown eye melanin did not produce significant cell lysis. The electron spin resonance (ESR) signals of blue and brown eye melanins were very similar to those of eumelanin. Comparison of these findings with our previous results indicated that the blue and brown eye melanins are essentially eumelanin. The ESR signals further suggested that in the case of both blue and brown eye melanins the iris, ciliary body, choroid, and retinal pigment epithelium did not differ.

Oxidase electrode measurements have shown that the neurotoxin metabolite 1-methyl-4-phenyl-2,3-dihydropyridinium autoxidizes to hydrogen peroxide and 1-methyl-4-phenylpyridinium in a reaction promoted by iron chelates. The mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity is discussed in the light of these findings.

Kinetics of growth and decay of triplet states of molecules at known orientation in a magnetic field have been measured. Amplitudes of the zero field eigenstates in each of the eigenstates at finite field were varied by choice of orientation. No interference between the amplitudes of the zero field states was found in the observed kinetics. It is concluded that excitations into the individual zero field components proceed via independent channels.

Free radicals produced during the autoxidation of 3,4-dihydroxyphenylalanine (DOPA) and other catechol(amine)s to melanins have been studied using electron spin resonance spectroscopy. Magnetic parameters for the radical intermediates have been determined, allowing the radicals to be unambiguously identified. Three types of radical are formed: the primary radical from one-electron oxidation of the parent catechol(amine); and two secondary radicals, one formed via OH- substitution, the other via cyclization. The formation of these radical species can be linked to molecular products formed during catecholamine oxidation and melanin formation.

The characterization and identification of semiquinone radicals from catechol(amine)s and catechol estrogens by electron spin resonance spectroscopy is addressed. The use of diamagnetic metal ions, especially Mg2+ and Zn2+ ions, to detect transient semiquinone radicals in biological systems and to monitor their reactions, is discussed. A brief account of the identification and reactions of quinones is also presented.

Synthetic dopa melanin and cysteinyldopa melanin have different electron spin resonance spectra. Data are reported for mixtures of these melanins and for dopa-cysteinyldopa copolymers, which are spectroscopically similar. A simple parameterization of the spectra allows estimation of the relative amounts of (i) dopa melanin and cysteinyldopa melanin in mixtures and of (ii) dopa and cysteinyldopa incorporated into copolymers. Several natural eumelanins and pheomelanins have been characterized and shown to be copolymers.

Oxidase electrode measurements as well as optical and electron spin resonance spectroscopic data have shown that synthetic neuromelanin oxidizes the neurotoxin metabolite 1-methyl-4-phenyl-2,3-dihydropyridinium in a dose-dependent manner forming 1-methyl-4-phenylpyridinium and hydrogen peroxide. Hydroxyl radicals are formed in this reaction which is promoted by iron chelates. In contrast, neither 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine nor 1-methyl-4-phenylpyridinium reacts with synthetic neuromelanin in a similar fashion. The mechanism of selective toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in pigmented neuronal cells is discussed in the light of these findings.

Electron spin resonance spectroscopy has recently been used by others to detect directly radical species in isolated perfused hearts. Sample processing prior to spectroscopy in this study involved pulverization of tissue, which can artifactually generate radical species. We assessed in isolated perfused hearts the influence of tissue pulverization on the identity of radical species detected by spectroscopy and then, using a processing technique less likely to induce artifacts, whether myocardial ischemia and reperfusion generate radical species. Rat and rabbit hearts (n = 8) were perfused aerobically for 10 min and freeze-clamped to -196 degrees C. Frozen tissue was processed at -196 degrees C for spectroscopic analysis by pulverization vs. chopping. Spectra of pulverized tissue consisted of three components: a semiquinone (g = 2.004), a lipid peroxy radical (g [ = 2.04 and g = 2.006), and a carbon-centered radical that is possibly a lipid radical (giso = 2.002 and AHzz approximately equal to 50 G). Chopped tissue consisted of a single component, a semiquinone (g = 2.004). Rat hearts (n = 8 per group) also underwent 10-min global no-flow normothermic ischemia followed by 5-60 sec of either aerobic or anaerobic reperfusion, with frozen tissue chopped prior to spectroscopy. Spectra of ischemic tissue consisted of an iron-sulfur center and a semiquinone. Aerobic reperfusion resulted in a spectrum similar to the control but with increased amplitude that peaked after 10-15 sec of reflow. Anaerobic reperfusion yielded a spectrum identical to that of ischemic tissue. We conclude that pulverization of frozen myocardial tissue arti-factually generates radical species. Using a nonpulverization technique for tissue processing, we found that myocardial ischemia and reperfusion produce radical species but that molecular oxygen is necessary for the burst of radical production during reflow.